Unraveling the genomic secrets of Tritonibacter mobilis AK171: a plant growth-promoting bacterium isolated from Avicennia marina

The scarcity of freshwater resources resulting in a significant yield loss presents a pressing challenge in agriculture. To address this issue, utilizing abundantly available saline water could offer a smart solution. In this study, we demonstrate that the genome sequence rhizosphere bacterium Tritonibacter mobilis AK171, a halophilic marine bacterium recognized for its ability to thrive in saline and waterlogged environments, isolated from mangroves, has the remarkable ability to enable plant growth using saline irrigation. AK171 is characterized as rod-shaped cells, displays agile movement in free-living conditions, and adopts a rosette arrangement in static media. Moreover, The qualitative evaluation of PGP traits showed that AK171 could produce siderophores and IAA but could not solubilize phosphate nor produce hydrolytic enzymes it exhibits a remarkable tolerance to high temperatures and salinity. In this study, we conducted a comprehensive genome sequence analysis of T. mobilis AK171 to unravel the genetic mechanisms underlying its plant growth-promoting abilities in such challenging conditions. Our analysis revealed diverse genes and pathways involved in the bacterium’s adaptation to salinity and waterlogging stress. Notably, T. mobilis AK171 exhibited a high level of tolerance to salinity and waterlogging through the activation of stress-responsive genes and the production of specific enzymes and metabolites. Additionally, we identified genes associated with biofilm formation, indicating its potential role in establishing symbiotic relationships with host plants. Furthermore, our analysis unveiled the presence of genes responsible for synthesizing antimicrobial compounds, including tropodithietic acid (TDA), which can effectively control phytopathogens. This genomic insight into T. mobilis AK171 provides valuable information for understanding the molecular basis of plant-microbial interactions in saline and waterlogged environments. It offers potential applications for sustainable agriculture in challenging conditions. Supplementary Information The online version contains supplementary material available at 10.1186/s12864-024-10555-0.


Background
Several methods have been attempted to address agricultural salinity challenges, such as developing salt-tolerant crops, soil amendments, crop rotation, and conventional breeding.Unfortunately, these approaches have proven inadequate, labor-intensive, and time-consuming.Consequently, it is imperative to implement alternative technologies to bolster agricultural sustainability, including using plant growth-promoting bacteria (PGPB) [1] with special emphasis on root microbiota.The root-associated biome comprises diverse microbial communities and blueprints the natural relationship between plants and microbes [2,3].Recently, several studies have demonstrated the efficacy of PGPB as modulators of salinity in plants and promoters of plant growth [4,5].Several mechanisms have been identified as contributing to stress mitigation, including biofilm formation, extracellular polymeric substances (EPS) production, nitrogen fixation, phytohormone production, and modulation of several plant's hormonal pathways [6][7][8].PGPB might induce the biosynthesis of several osmoprotectants, like trehalose, proline, glycine, phenols, and flavonoids, that reduce the reactive oxygen species (ROS) and reactive nitrogen species (RNS) in cells [Gupta, 2022[Gupta, #2511] ] [Kurniawan, 2021 #2513].In addition, terpene-producing PGPR may support crop plant growth and yield [Oleńska, 2020[Oleńska, #2517]].Such as Bacillus and Pseudomonas, which increase the yield of apples, tomatoes, and peppers [Katsenios, 2021 #2518].The regulation of terpene biosynthesis may enhanve the tolerance of cotton submergence under soil flooding [Sun, 2023[Sun, #2516]].
The genomes of salt-tolerant bacteria and archaea found in marine environments offer a wealth of information about their unique adaptations to survive in extreme conditions.Several genomes of salt-tolerant bacterial and archaeal strains of the marine environment have been completed for over 44 taxa, including Alphaproteobacteria such as Marinobacter and Roseobacter [9], revealing a biodegradable potential in high salinity environments.The Roseobacter group is adapted to the marine environment [10] and comprises chemotrophic bacteria often associated with eukaryotes [11].Members of this group have a much higher metabolic and ecological versatility than other dominant marine bacteria [12].Producing compatible solutes such as choline, ectoine, and hydroxyectoine also enhances the bacteria's ability to tolerate stresses such as salinity or high temperature [Vargas, 2008[Vargas, #2519]].
Tritonibacter mobilis is an alpha-proteobacterium from the abundant Roseobacter clade [13].It is formerly known as Ruegeria mobilis [14], and R. pelagia [15,16], representing 0.2% of the bacterial population in the surface ocean [17].Nevertheless, R. mobilis exhibits extensive genome divergence and evolutionary events leading to the speciation of marine bacteria, where horizontally transferred genes mostly originate from bacteria of the Roseobacter group.In addition, R. mobilis has been shown to produce the antibiotic tropodithietic acid (TDA) as a multifunctional secondary metabolite.TDA is a weak iron-chelating [18] agent with hormetic effects on nearby organisms, such as antibiosis [19].This bacterium is adapted to a surface-attached lifestyle [20], as one-third of TDA-producing bacteria are host-associated [21].
The marine environment is home to diverse microorganisms, many of which have evolved unique adaptations to survive in highly saline conditions.Salt-tolerant bacteria and archaea are particularly interesting to scientists studying extremophiles and their potential for industrial and biotechnological applications.Here, we present the complete genome sequence of the PGPB Tritonibacter mobilis AK171, isolated from the rhizosphere of mangroves.AK171 has salinity stress tolerance growthpromoting abilities on Arabidopsis thaliana.It provides new tools for enhancing our understanding of PGPR and applications in sustainable agriculture systems and a gold mine for several metabolites with potential industrial applications.

The isolation and growth conditions of AK171
AK171 was isolated from the rhizoplane of the intertidal plant A. marina growing in the coastal area of the Red Sea (GPS: 22.339914°N, 39.087972°E), Thuwal, Saudi Arabia, as described previously [22].In brief, the roots of A. marina were collected, and the excess soil was manually removed by shaking the roots and then placed in a sterile 0.9% phosphate-buffered saline (PBS) solution and vortex for 15 s.The cleaned root was placed sonicated for 30 s at 50-60 Hz.The liquid PBS "rhizoplane" compartment was serially diluted, plated on the filtered seawater yeast agar (FSY) supplemented with Luria-Bertani 35 gm/L (L7025, Sigma-Aldrich) and solidified with 2% Agar (SLB), and incubated at 30 °C until colony appears.AK171 was picked among > 300 isolates; based on initial identification and biochemical screening, AK171 was selected for further analysis.The colonies were creamcolored, opaque, circular, and convex with entire edges.The pure culture of AK171 was then regularly growing on/in Zobell Marine 2216E (ZM) (Bio Basic Asia Pacific Ltd, Singapore) at 30 °C.

Biofilm formation assessment
Pure cultures of AK171 were grown overnight in ZM broth (Lennox L Broth Base, Invitrogen) at 30 °C without shaking for 16 h.Fresh cultures were prepared by inoculating 3 mL ZM media with 100 µL of the pre-culture and allowed to grow for 4 h until reaching the exponential phase.A total of 20 µl of the exponential culture was spotted on Congo red (CR) plates (1% tryptone, 1% agar, 20 µg/mL Congo red, and 10 µg/mL Coomassie brilliant blue G250) [23].Plates were incubated at 30 °C for up to 7 days to evaluate colony morphology and color.The quantitative assessment was done using the microtiter plate method (MTP) by crystal violet, as described previously [25].200 µl of ZM media was inoculated in wells of sterile 96-well microtiter polystyrene tissue culture plates.After cultivation for 24 h at 28 °C, the well contents were discarded and washed 3x with 200 µl sterile phosphatebuffered saline PBS (pH 7.2).The attached cells were precipitated with 200 µl of Sodium acetate (2%) and incubated for 5 min, then washed once with ddH 2 O.The biofilm was stained by adding 200 µl of crystal violet (0.1%) for 30 min at room temperature.The stained biofilm was washed 3x with ddH 2 O before diluting in 33% acetic acid and then measured the OD at 630 nm.OD 630nm < 0.120 was considered absent or weak biofilm, OD 630nm 0.120-0.240as moderate biofilm, and OD 630nm > 0.240 as a strong biofilm.

Arabidopsis salt stress tolerance assays
A. thaliana Col-0 seeds were surface sterilized for 10 min in 70% ethanol (v/v) solution supplemented with 0.05% triton-X, then washed 4 times with 100% ethanol.The seeds were left to dry in aseptic conditions on sterilized filter paper until use.Seed colonization with bacteria and plant growth conditions was conducted as described previously [26].For the inoculation procedure, 100 µL of the bacterial culture (10E8 cells) mixed with 50 mL of the half-strength Murashige and Skoog basal medium (MS) (Sigma-Aldrich, St. Louis, MO, USA) (pH 5.8, adjusted with 10 mM KOH) and solidified with 0.9% Agar (Sigma-Aldrich).After that, five days-old seedlings with root lengths ~ 1-1.5 cm (6 seedlings/plate) were aseptically transferred using sterile toothpicks to the normal and stress plates.The first was the Standard plate Method (SPM), in which total agar medium in 90 mm square plates was used either filled with ½ MS as normal conditions or ½ MS stratified with 100 mM NaCl (5.844 g/L) as salt stress conditions.In the control (MOCK) with noninoculated seeds, 100 µl SLB liquid medium was mixed with ½ MS medium.A Waterlogging Method (WM) was used to mimic the hypoxic stress conditions and salt stress conditions naturally occurring in the mangrove ecosystem.In this method, five-day-old seedlings were transferred on the top of ½ MS agar blocks without salt in normal conditions or amended with 100 mM NaCl in salinity conditions.The discs were also submerged in ½ MS liquid media supplemented with the same salt concentration, either none in normal (0 mM NaCl) or salinity conditions (100 mM NaCl).The plates of both methods were incubated in Percival with a light intensity of 150-200 µmol m − 2 s − 1 in 16 h light/8 h dark photoperiod at 22 °C for 15 days.Then, the total fresh weight (FW) was recorded to evaluate the effect of the bacterial treatment on the plant growth under both conditions.

Biocontrol assessment against potential phytopathogens
The antagonistic effect of T. mobilis AK171 was assessed in vitro against a virulent strain of the plant pathogen Pseudomonas syringae pv.tomato DC3000 (Pst).In vitro testing was conducted by growing the tested bacteria (OD600nm = 0.2) on King's B medium in an orbital shaker (120 rpm) at 28 °C.A mixture of 25 µL of this culture with 25 mL of cooled water yeast agar was prepared.After the agar solidified, 10 µL of AK171 (OD600nm = 0.2) was added as replicates of the antagonistic bacteria, with a spacing of 3.5 cm on a 9 mm petri dish.The plates were then incubated at 30 °C for 24-48 h, and the appearance of an inhibition zone was considered a positive result.
For the in vivo test, 100 µl of overnight culture of both the antagonistic bacteria (T.mobilis AK171) and the tested bacteria (Pst DC3000), were refreshed in 5 ml of their preferred media to reach the exponential phase.An amount of 50 mL of each bacterium was then adjusted to OD 600nm 0.2 and mixed with 50 mL ½ MS Agar.Then, surface sterilized seeds of A. thaliana Col-0 were sown on the solidified agar and transferred to Percival incubators within the same conditions of plant assay mentioned previously for 15 days.The survival rate was calculated compared to the negative control (survival value 20%) of Col-0 colonized with the tested bacteria alone (Pst, the pathogenic/tested strain).As a positive control, Pseudomonas fluorescens L111 was used as the antagonist strain (survival value > 40%).L111 was provided by Prof. David Dowling, Institute of Technology Carlow, Carlow, Ireland.We used Col-0 without colonization (MOCK) and Col-0 plants inoculated with AK171 alone as controls.

Genomic DNA extraction
Total genomic DNA was extracted from a pure culture of AK171 using the GenElute™ Bacterial Genomic DNA Kit (Sigma Aldrich, Germany) according to the manufacturer's instructions.DNA integrity, quality, and quantity were assessed by agarose gel electrophoresis 1%, Nano-Drop 2000 spectrophotometer (Thermo Fisher Scientific, Schwerte, Germany), and the concentration by Qubit dsDNA high-sensitivity (HS) Kit (Thermo-Fischer Scientific).

Phylogenomic classification of AK171
For whole genome-based taxonomic analysis, genome sequence data were uploaded to the Type Strain Genome Server (TYGS) (https://tygs.dsmz.de).Two complementary ways were used to determine the closest type-strain genome.First, the AK171 genome was compared to genomes of all type strains available in the TYGS database via the MASH algorithm, a fast approximation of intergenomic relatedness, and the 10 type strains with the smallest MASH distances were chosen.Second, an additional set of 10 closely related type strains was determined via the 16 S rDNA gene sequences.These were extracted from the user genomes using RNAmmer [35].Each sequence was subsequently BLASTed against the 16 S rDNA gene sequence of the currently 18,361-type strains available in the TYGS database.This was used as a proxy to find the 50 best-matching type strains (according to bitscore) for the AK171 strain genome and subsequently calculate precise distances using the Genome BLAST Distance Phylogeny (GBDP) approach under the "coverage" algorithm and distance formula d5 [35].These distances were finally used to determine the 10 closest type strain genomes to the AK171.For the phylogenomic inference, all pairwise comparisons among the set of genomes were conducted using GBDP, and accurate intergenomic distances were inferred under the algorithm 'trimming' , and distance formula d5 and 100 distance replicates were calculated each.Digital DDH values and confidence intervals were calculated using the recommended settings of the GGDC 3.0.The resulting intergenomic distances were used to infer a balanced minimum evolution tree with branch support via FASTME 2.1.6.1, including SPR postprocessing [45].Branch support was inferred from 100 pseudo-bootstrap replicates each.The trees were rooted at the midpoint and visualized with PhyD3 [46].Furthermore, Orthologous Average Nucleotide Identity Tool (OAT) software [47] calculated Ortho-ANI, ANIb, and ANIm values between the close strains of the AK171.Average Amino Acid Identity (AAI) was performed using the Java program EzAAI [48].

Statistical analyses
The data from the plant screening assay were subjected to non-parametric one-way ANOVA or the Kruskal-Wallis test [49].The statistical difference is based on the Paired t-test/ Dunn's multiple comparisons tests.All statistical analysis was done using GraphPad Prism version 9.5.0 (525) software (https://graphpad.com).

Data deposition
The genome sequence of Tritonibacter mobilis AK171 has been submitted to the NCBI GenBank database under accession number CP126134 in BioProject no.PRJNA973967.

Morphological and plant growth promotion characteristics
Tritonibacter mobilis AK171 colonies have a circular, cream-colored phenotype and measure 2 mm in diameter.The colonies turn brown after 24 h when growing on ZM agar at 30 °C.The cells were rod-shaped (length: 3-5 µM), forming a rosette arrangement as found by SEM (Fig. 1).AK171 can tolerate heat stress for up to 37 °C and grow well under saline conditions up to a concentration of 2 M NaCl (Supplement Table S1).The qualitative evaluation of PGP traits showed that AK171 could produce siderophores and IAA but could not solubilize phosphate nor produce hydrolytic enzymes (Supplement Table S1).

Biofilm formation, plant growth promotion, and salinity stress alleviation
The qualitative biofilm test showed changes in the color of CR media, as shown in Supplement Figure S1-A, while the qualitative test resulted in OD 630nm = 0.24, indicating a moderate capacity to form biofilms.In planta, T. mobilis AK171 enhanced A. thaliana growth under both normal (½ MS) and salt stress conditions (½ MS + 100 mM NaCl).
After 15 days of growth, A. thaliana seedlings treated with AK171 showed bigger shoot and root systems and an increment of > 25% in fresh weight compared with non-inoculated control plants (Fig. 2A) in both methods.Under the SPM method, AK171 increased the fresh weight of the seedling biomass of 20-day-old seedlings under normal and salinity-stressed conditions (Fig. 2B) by 27.8% and 33.6%, respectively.Using the WM method, the fresh weight measurements for 20-day-old seedlings exhibited similar increases under both normal and salinity-stressed conditions (35.2% and 47.3%, respectively) (Supplementary Table S2).

Biocontrol potential of Tritonibacter mobilis AK171
In vitro experiments, it was observed that the growth of Pseudomonas syringae pv.tomato DC3000 (Pst) was inhibited, and a clear zone devoid of growth was visible around the T. mobilis AK171.This indicates that AK171 has the ability to suppress the growth of Pst in vitro.Furthermore, the results obtained from the in vivo assessment revealed a significant enhancement in the survival rate of A. thaliana when AK171 was introduced during the challenge with Pst.Particularly, the survival rate of A. thaliana increased by more than 50% when exposed to Pst in the presence of AK171, compared to the negative control, which employed only the pathogenic Pst strain.These findings provide compelling evidence that the incorporation of T. mobilis AK171 exerts a pronounced positive influence on the survival of A. thaliana in the presence of the pathogenic Pst strain (Supplementary Fig. 1-B).
By performing genome analysis using antiSMASH, AK171 was found in Figure S2 to harbor five gene clusters.These clusters include two gene clusters responsible for the biosynthesis of homoserine lactone and one gene cluster for beta lactone biosynthesis, exhibiting 13% similarity to corynecin I, II, and III.Additionally, AK171 possesses an ectoine biosynthetic gene cluster with 100% similarity to ectoine.Furthermore, there is a Type I polyketide synthase (PKS) biosynthetic gene cluster and an NPRS-like biosynthetic gene cluster, which do not exhibit similarity to any known clusters.
However, to check the reliability of evolutionary distance assessment between bacterial species based on digital whole genome comparison, average nucleotide identity (OANI, ANIb, and ANIm) was also measured.Five complete genomes of closely related AK171 genome were retrieved from the NCBI GenBank database and Orthologous Average Nucleotide Identity Tool (OAT) software.Their relationships and evolutionary distance were assessed based on ANI values.As shown in (Fig. 6, Table S3B, C and D) OrthoANI, ANIb, ANIm have a value of > 95% obtained with Tritonibacter mobilis.Identity at the level of amino acid also shows a high AAI percentage of > 98% with Tritonibacter mobilis (Supplementary Table S3E, Fig. 7).

Adaptation of AK171 to stress conditions
The adaptation strategy of bacteria to salt stress conditions is multifaceted, involving the regulation of osmotic pressure, modifications in cell membrane composition, ion transport mechanisms, and the activation of stress response pathways.These adaptive mechanisms enable bacteria to survive and thrive in salt-rich environments by maintaining cellular homeostasis and protecting essential cellular components from the detrimental effects of high salt concentrations [51].The growth of AK171 in highly saline media indicates that AK171 is a moderately halophilic bacterium that can tolerate NaCl concentrations up to 2 M (Supplementary Table S1).This indicates the ability to tolerate salt stress contributes to the survival of AK171 in the coastal intertidal plant environment of mangroves (A.marina) and the Red Sea.One primary mechanism through which bacteria adapt to high salt conditions is by regulating their internal osmotic pressure.They do this by accumulating or synthesizing compatible solutes, such as amino acids, sugars, and polyols, which help balance the osmotic pressure between the inside and outside of the cell.These compatible solutes act as osmoprotectants and prevent dehydration and shrinkage of the bacterial cells in hypertonic environments [52].Genome mining of AK171 revealed the presence of genes related to the production of osmoprotectants, osmoregulation, and abiotic stress tolerance, such as heat, acidity, and oxidative stress (Supplementary Table S7).In addition, the superoxide dismutase (Sod2) gene (AK171_GM001633) can protect bacterial cells from oxidative stress, and two genes of KatG-producing  S7).Many studies directly elucidated that microorganisms accumulate polyhydroxyalkanoate (PHA) as an intracellular metabolic storage component, providing microorganisms with higher stress survival and strength [54].A large number of polyhydroxyalkanoate (PHA) genes were found in the genome of the AK171 (AK171_GM001104, AK171_GM001105, AK171_GM001106, AK171_GM001107, AK171_ GM001108, and AK171_GM001109).
Ectoine is a potential enzyme protector against stress as heat, cold, and high or low pH [57].Several genes of action were found, including (Supplementary Table S7); ectA gene (AK171_GM002074) coding L-2,4-aminobutyric acid acetyltransferase, ectB gene (AK171_GM002073) coding for dat diamino butyrate-2-oxoglutarate transaminase and ectC gene (AK171_ GM002072) coding for L-ectoine synthase were found.The presence of ectoine, an osmoprotectant compound, in the genome of AK171 might aid in its tolerance to salinity in its environment.In addition, ectoin-producing bacteria stimulate the root growth of treated seedlings under salinity stress [57].
Moreover, apart from synthesizing various osmoprotectants, carotenoid production has been identified as crucial for bacterial survival in the rhizosphere.Carotenoids also play a significant role in shielding bacteria against UV radiation and fostering the bacterial-plant interaction [58].Pantoea sp.YR343 ΔcrtB mutant, which lacks the phytoene synthase gene (crtB), served to underscore the significance of carotenoids in facilitating a successful plant-microbe interaction.As a result of the mutation, this strain was unable to produce carotenoids, suggesting the role of carotenoids in plant colonization.The genome mining revealed a large number of carotenoid biosynthetic genes this including (AK171_ GM000050, AK171_GM000066, AK171_GM000257, AK171_GM000454, AK171_GM000487, AK171_ GM000504, AK171_GM000541, AK171_GM000634, AK171_GM000882, AK171_GM000897, AK171_ GM000954, AK171_GM001425, AK171_GM001509, AK171_GM001638, AK171_GM001720, AK171_ GM002291, AK171_GM002306, AK171_GM002466, AK171_GM002651, and AK171_GM002928).
The ubiquitin (UBI) ubiEGH genes are involved in the terpenoid biosynthesis pathway and Volatile Organic Compounds (VOCs) antagonism [59]; in AK171, several genes, including (AK171_GM000214, AK171_ GM002976, AK171_GM002851, AK171_GM000066, and AK171_GM000487) were found.A novel gene cluster (moc) associated with E1 catabolism was predicted in AK171 (Supplementary Table S7).The 3-hydroxybenzoate 4-monooxygenase (mocA; a single-component flavoprotein monooxygenase) identified in the moc cluster was found to be necessary, such as the gene mocA and of AK171 bacteria (AK171_GM001034).moaA (AK171_ GM001881) and moaC for (cPMP synthase) (AK171_ GM001466).These genes facilitated the biosynthesis of molybdenum cofactor (mocO) [60].The adaptation strategies of AK171 to abiotic stress conditions are diverse and involve the production of stress proteins, modifications in cell membranes, osmoprotectant synthesis, pH homeostasis, and detoxification mechanisms.These strategies enable AK171 to survive and thrive in challenging environments by maintaining cellular homeostasis, reducing cellular damage, and preserving essential functions.

AK171 is a potential PGPR for applications
Plant growth-promoting rhizobacteria hold great potential for various agricultural and environmental applications.Their unique capabilities stimulate plant growth and enhance plant health, making them valuable assets in sustainable agriculture and ecosystem management.One key application of PGPR is its ability to improve crop productivity.By enhancing nutrient availability and uptake, fixing atmospheric nitrogen, promoting root development, producing growth-promoting hormones, and protecting plants against pathogens and abiotic stresses.These beneficial effects contribute to increased crop yields, reduced reliance on chemical fertilizers, and improved soil fertility [61].AK171 exhibited several plant growth-promoting traits involved in nutrient acquisition and modulating plant hormone levels that could possibly be responsible for growth promotion.The ability to produce IAA gives AK171 a potential application as PGPR.Several tryptophan biosynthesis pathway trpCDEGS genes were identified in AK171, including (AK171_ GM001465, AK171_GM001463, AK171_GM001460, AK171_GM001462, AK171_GM000409, and aroCEF genes including (AK171_GM002695, AK171_GM002805, and AK171_GM000228) they are involved in synthesis and transporter of IAA (Supplementary Table S7).IAA is the most common auxin, which is crucial in developing the embryonic pattern, vascular tissues, leaf, secondary roots, flower initiation, and stem cell maintenance [62].IAA was found to improve the development of rice deep root systems under stress conditions [63].Bacteria use various mechanisms to compete for iron, an essential element that can limit their growth.These include specific iron uptake transporters, the secretion of diverse siderophores, and the synthesis of siderophore receptors to utilize siderophores from other microorganisms.A strong iron uptake system also helps protect host plants from pathogens by limiting the iron available to the siderophores [64].AK171 genome mining revealed around 29 genes involved in iron-and heme-uptake.Among them, several iron ABC transporters: AfuABC (AK171_GM001781, AK171_GM001782, and AK171_ GM001783), as well as six genes coding for the iron complex-outer membrane receptor, MFS transporter and several ferrous iron uptake transporters system and 4 genes for heme exporter ccmABCD (AK171_GM001605, AK171_GM001606, AK171_GM001607, and AK171_ GM001608) (Supplementary Table S5).The presence of efficient Fe-uptake systems can help AK171 compete for Fe in such environments.
Salicylate biosynthesis protein (AK171_GM002244) is involved in the biosynthesis of pyochelin, a siderophore molecule for iron acquisition [65].As well as other siderophore genes including ABC-type Fe 3+ -siderophore transport system COG0609 (AK171_GM000303, AK171_GM001323, AK171_GM002562, and AK171_ GM002887) and COG1120 ABC-type cobalamin/Fe 3+siderophores transport system (AK171_GM001324, AK171_GM002563, and AK171_GM002888) were also found in the genome of T. mobilis AK171 (Supplementary Table S5).This suggests that AK171 is not only able to solubilize Fe but can also import and export it to the host plant.
In fact, R. mobilis was reported as a producer of TDA as a sulfate-containing compound with iron-chelating properties that may contribute to other functions, such as quorum sensing [18] and biofilm formation [68].In particular, unlike siderophores, bioactive TDA is typically upregulated in the absence of iron.The weak iron-chelating properties of TDA indicate that iron sequestering is not its main function but is potentially related to its mode of action [69] or to symbiosis where the TDA-iron complex could serve as an iron reservoir [70].This is also supported by a previous study of the Roseobacter group that metabolizes dimethylsulfoniopropionate (DMSP) [71].In addition, TDA-producing Tritonibacter can catalyze DMSP and might produce dimethyl sulfoxide (DMS), which then serves as a source of sulfur or an anti-stress component that readily scavenges hydroxyl radicals and other reactive oxygen species [72].
Overall, the unique attributes and functions of AK171 make them valuable allies in sustainable agriculture, disease management, environmental remediation, and promoting plant health.Harnessing the potential of PGPR offers promising avenues for addressing agricultural and environmental challenges more efficiently, eco-friendly, and sustainably.

AK171 chemotaxis, biofilm formation, and plant colonization
Many plant-associated bacteria are crucial in adhering to and colonizing plant roots.These bacteria produce several substances, including polysaccharides such as 3-deoxy-manno-octulosonate cytidyl (CMP), to facilitate this process.These contribute to the formation of biofilms, which serve as protective barriers and allow the bacteria to establish a stable presence on the root surface.The production of cellulose and exopolysaccharides by plant-associated bacteria is an important mechanism that enables them to interact with plants and establish beneficial relationships [73].EAL (Glu-Ala-Leu) and GGDEF (Gly-Gly-Asp-Glu-Phe) domain proteins are common in bacteria and regulate biofilm formation.Diguanylate cyclases DGCs synthesize c-di-GMP, a key signaling molecule, while PDEs break it down.c-di-GMP controls the transition between biofilm and planktonic growth.High levels of c-di-GMP promote biofilm formation, while low levels favor planktonic growth.DGCs produce c-di-GMP by converting GTP, and PDEs degrade c-di-GMP into GMP.The balance between DGCs and PDEs is crucial for regulating bacterial growth lifestyles, especially in biofilm formation.[68].In AK171, genome mining revealed the presence of 16 genes coding the EAL and GGDEF domain protein genes and diguanylate cyclases and phosphodiesterases.This includes AK171_GM000451, AK171_GM000620, and AK171_ GM001571 coding GGDEF domain; AK171_GM000250, AK171_GM000555, and AK171_GM001049 coding EAL, and GGDEF domains; AK171_GM000964, and AK171_GM001576 coding c-di-GMP domain; AK171_ GM000450, AK171_G000479, AK171_GM000555, AK171_GM000615, AK171_GM000620, AK171_ GM001049, AK171_GM001229, and AK171_GM002786 coding diguanylate cyclases, and AK171_GM000677, AK171_GM000878, AK171_GM000964, AK171_ GM000972, AK171_GM001048, AK171_GM001075, AK171_GM001098, AK171_GM001570, AK171_ GM001576, AK171_GM001775, AK171_GM002311, AK171_GM002631, and AK171_GM002759 coding phosphodiesterases.In summary, in AK171, the EAL and GGDEF domain protein genes encode DGCs and PDEs, respectively, and their enzymatic activities are central players in regulating bacterial biofilm formation by modulating the levels of c-di-GMP.These mechanisms influence the behavior and characteristics of bacterial biofilms, including adhesion, colonization, matrix production, and motility.
In addition, the presence of type IV pili (T6P) in bacterial species has been closely associated with biofilm formation.These appendages aid in adhesion, provide structural support, enable interbacterial interactions, and facilitate bacterial colonization within the biofilm.AK171 has all genes essential for the formation, regulation, and assemble of the Type VI system, including AK171_ GM002090, AK171_GM002091, AK171_GM002093, AK171_GM002097, AK171_GM002098, AK171_ GM002099, AK171_GM002100, AK171_GM002103, and AK171_GM002104 (supplementary table S4).Bacterial biofilms can be regulated by a mechanism by which small signaling molecules called autoinducers are used for cellular communication, called quorum sensing (QS), allowing bacteria to regulate gene expression in a cell-density-dependent manner [74,75].The genome of AK171 contains genes for colonization, root surface adhesion, biofilm formation, quorum sensing, and secretion system (Supplementary Table S4).Understanding the role of T6P in biofilm development is crucial for deciphering the complex dynamics and behavior of bacterial biofilms, which can have significant implications in various fields, including medicine, industry, and environmental sciences.
In parallel, the marine Roseobacters representative genus, Phaeobacter inhibens, was studied in several aquaculture systems and is known for its potential to produce siderophores and acylated homoserine lactones (AHL).One gene (AK171_GM000963) was involved in N-acyl-L-homoserine lactone synthetase, and one gene (AK171_GM001294) coding Acyl-homoserine lactone (AHL) acylase was identified.The rhtB genes encoding a homoserine/homoserine lactone efflux protein were found in the AK171 genome (AK171_GM000078) and mlhB, chnC genes encoding epsilon-lactone hydrolase (AK171_GM000417) (supplementary Table S7).Similar proteins were found in the antiSMASH results of the AK171 genome as homoserine lactones and beta lactones.These acylated homoserine lactones are pivotal in quorum sensing and antibacterial agents [76].
Rhodobacterales (Roseobacter clade), including aerobic anoxygenic phototrophic (AAP) bacteria, represent an important part of marine microbial communities [78] and are thought to be important for the carbon cycle of the ocean by harvesting light by bacteriochlorophyll (BCl) and carotenoids.It could be investigated by looking for the photosynthetic gene cluster (PGC) in AK171.A key enzyme in this pathway, including the light-independent chlorophyllide reductase bchF (AK171_GM001606), the phosphorus metabolism AMGs [79] genes pstS (AK171_ GM002863), and phoU (AK171_GM001354), and Protochlorophyllide reductase (AK171_GM000352).These proteins enable a broad view of the phylogeny of anoxygenic photosynthetic bacteria with a capacity to synthesize bacteriochlorophyll [80].
Bacterial dual lifestyle as free living in soil or association with plant roots provides access to various essential nutrients for bacterial proliferation and survival.Therefore, the genome of AK171 is equipped with a multitude of genes involved in the uptake, transport, and metabolism of nitrogen, sulfur, and carbon-based compounds.The list of metabolites and corresponding encoding genes, e.g., ABC transporters, Major Facilitator Superfamily (MFS) transporters, and Phosphotransferase systems (PTS), are listed in Supplement Table S5.
A large number of genes (72) encoding two-component systems (TCS) for rapid sensing and adjustment to changes in the external environment were present in AK171.The TCSs (two-component systems) in AK171 belong to several families, including OmpR, NtrC, NarL, and CheB/CheR.These TCSs play important roles in various cellular processes, e.g. in the phosphate starvation phoBR and phoPQ and the production of acid phosphatases.They regulate the cell's response to phosphate limitation and aid in the production of acid phosphatases [84,85].Also in nitrogen metabolism, including glnGL, glnKR, and narXL.They play roles in the assimilation and utilization of nitrogen by the bacterial cell [3] the complete list of TCS is in supplementary Table S6.These mechanisms enable AK171 to find optimal growth conditions, enhance survival and resistance, acquire nutrients, establish beneficial relationships, and respond to environmental changes, ensuring their successful adaptation and colonization.

AK171 bioactive secondary metabolites
Bioactive secondary metabolites produced by bacteria play a significant role in their adaptation and survival.These metabolites possess diverse biological activities, including antimicrobial, antifungal, and antiviral.One of the primary benefits of bioactive secondary metabolites for bacteria is competitive advantage.These compounds can inhibit the growth of other microorganisms, providing protection and resource competition.Bacteria can secure their niche in a specific environment by producing bioactive metabolites and fending off potential competitors [86].The genome mining of AK171 reveals the presence of several operons of secondary metabolites; this includes Type I polyketide synthase (PKS) gene clusters such as T1PKS, terpene, and non-ribosomal peptide synthetases (NPRS-like) [87].In bacteria the type 1PKS are novel and composed of modules that further consist of multiple domains covalently linked in a very long polypeptide to catalyze specific reactions, including the mini-PKS consisting of ketosynthase (KS), acetyltransferase (AT), and ACP and the accessory domains such as ketoreductase (KR), dehydratase (DH), enoyl reductase (ER), and methyltransferase [88].T1PKS/ nonribosomal peptide synthetase (NRPS) hybrid BGC was found in the clusters resulting from antiSMASH.Two atoB genes coding for acetyl-CoA C-acetyltransferase (AK171_GM000076 and AK171_GM001085).Two genes coding for cyclohexanone degradation (AK171_ GM002241 and AK171_GM001085).One gene murE coding for UDP-N-acetylmuramoyl-L-alanyl-D-glutamate-2,6-diaminopimelate ligase (AK171_GM000770).One gene pat coding for phosphinothricin acetyltransferase (AK171_GM000918).The PKS gene clusters have been distinguished as cis-acyltransferase (cis-AT) and trans-acyltransferase (trans-AT) based on the reliance of trans-AT PKS on separately encoded ATs for selecting α-carboxyacyl-CoA polyketide building blocks.Notably, the trans-AT PKS biosynthetic clusters are an evolving group of modular PKSs that are becoming more ubiquitously found in microbial genomes.In AK171, several genes encoding PKS synthesis, including lpxD coding for UDP-3-O-[3-hydroxymyristoyl] glucosamine N-acyltransferase (AK171_GM001394), plsC coding for 1-acyl-sn-glycerol-3-phosphate acyltransferase (AK171_GM001403), plsX coding for glycerol-3-phosphate acyltransferase (AK171_GM001700), lpxA coding for UDP-N-acetylglucosamine acyltransferase (AK171_GM001242), UDP-3-O-acylglucosamine N-acyltransferase (AK171_GM000918), yafP coding acetyltransferase (AK171_GM002634), COG0204 coding for 1-acyl-sn-glycerol-3-phosphate acyltransferase (AK171_ GM002267) and finally the ppsE coding for phthiocerol/ phenolphthiocerol synthesis polyketide synthase type I (Q7TXL6).
The Non-ribosomal Peptide Synthetases (NRPS) are large, multi-modular enzyme complexes involved in the biosynthesis of a diverse group of natural products known as non-ribosomal peptides (NRPs).NRPSs are found in bacteria, fungi, and plants and are crucial in producing bioactive compounds with various biological activities, including antimicrobial, anticancer, and immunosuppressive properties [87].In AK171, several genes were found related to NRPS, including genes encoding saccharopine dehydrogenase (NAD + , L-lysine-forming) (AK171_GM000120) and type I glyceraldehyde-3-phosphate dehydrogenase (AK171_GM000381) (Supplement Figure S2).In addition, the AK171 genome encodes for several NRPS-like genes involved in lysine biosynthesis (Supplement Table S7).Lysine is an essential amino acid required for protein synthesis and is crucial in cellular metabolism.While lysine biosynthesis in bacteria is typically achieved through a series of enzymatic reactions.These NRPS-like genes are often called Lysine-activating peptide synthetase (LAP) genes.LAPs are similar in structure to NRPSs, with domains responsible for activating and incorporating amino acids.However, unlike traditional NRPSs, LAPs primarily function in lysine synthesis.NRPS-like LAP genes associated with lysine biosynthesis are found in various bacterial species.For example, the dapX gene in Escherichia coli [89] encodes a LAP involved in lysine synthesis.Another example is the lysX gene in Bacillus subtilis [90], which encodes a LAP involved in lysine activation.NRPS-like genes for lysine biosynthesis suggest the versatility of bacterial metabolic pathways and highlight the diversity of mechanisms for producing essential metabolites like lysine.These genes provide insights into the adaptive strategies AK171 employs to produce lysine and overcome limitations in the availability of exogenous lysine sources (Supplement Table S7).
Through the modular nature of NRPS assembly, the combination and arrangement of specific domains within the NRPS complexes can be altered to produce different peptide products.Due to their importance in synthesizing bioactive compounds, enzymes have become a valuable target for drug discovery and engineering novel peptide-based therapeutics [87,91,92], which gives AK171 a new avenue for industrial applications.

Conclusions
The scarcity of freshwater resources and consequential yield losses pose significant challenges in modern agriculture.However, this study presents an innovative solution by utilizing Tritonibacter mobilis AK171, a halophilic marine bacterium capable of thriving in saline and waterlogged environments.The comprehensive genome sequence analysis of T. mobilis AK171 has shed light on the genetic mechanisms underlying its ability to adapt and thrive in salinity and waterlogging stress.Activating stress-responsive genes, producing specific enzymes and metabolites, and forming biofilms contribute to its remarkable tolerance to high salinity and waterlogging.Additionally, the presence of genes responsible for the synthesis of antimicrobial compounds, such as tropodithietic acid (TDA), unveils the potential for T. mobilis AK171 to effectively control phytopathogens, e.g., Pseudomonas syringae pv.tomato DC3000 is widely recognized for its ability to infect a diverse range of plant hosts, including various crops and ornamental plants.This antimicrobial activity further enhances its value for sustainable agriculture practices, creating eco-friendly alternatives to conventional chemical interventions.The findings from this study not only advance our understanding of plant-microbial interactions in saline and waterlogged environments but also provide promising opportunities for addressing the challenges of water scarcity and improving agricultural productivity in challenging conditions.By leveraging the genetic potential of T. mobilis AK171, sustainable and eco-friendly solutions can be realized, paving the way for a more resilient and efficient agricultural sector.Overall, this study highlights the enormous potential of halophilic marine bacteria and their ability to revolutionize agriculture in the face of water scarcity and environmental challenges.

Fig. 2 Fig. 1
Fig. 2 AK171 enhances salt stress tolerance of Arabidopsis.Fresh weight enhancement of Arabidobsis thaliana seedlings germinated with AK171 in 1/2MS and stress conditions 100mM NaCl compared to non-colonized plants (MOCK).It shows both the significance and the growth of 20-day-old seedlings.The seedlings were stressed in (A) a solid (aerated) phase and (B) in a submerged root environment.Bar, 2 cm

Fig. 5
Fig. 5 Phylogenomic classification of AK171 strain based on Whole Genome Sequence.Phylogenomic tree based on Genome Basic Local Alignment Search Tool (BLAST) distance phylogenies (GBDP) using Type Strain Genome Server (TYGS) platform

Fig. 7 Fig. 6
Fig. 7 Phylogenomic classification of AK171 strain based on Average Amino Acid Identity (AAI).The top five highly identical bacterial species were analyzed for AAI